Cell strains for use in identifying potentially therapeutically effective agents

ABSTRACT

The present invention provides cell strains suitable for use in screening agents for potential therapeutic efficacy. The cell strains retain responsiveness to apoptosis inducers and inhibitors.

This is a divisional of U.S. Ser. No. 08/366,368 filed Dec. 29, 1994.

FIELD OF THE INVENTION

The present invention is directed to methods to detect therapeuticallyeffective agents, particularly those that affect apoptosis, mutagenesis,cellular proliferation and cellular differentiation.

BACKGROUND OF THE INVENTION

Apoptosis is a normal physiologic process that leads to individual celldeath. This process of programmed cell death is involved in a variety ofnormal and pathogenic biological events and can be induced by a numberof unrelated stimuli. Changes in the biological regulation of apoptosisalso occur during aging and are responsible for many of the conditionsand diseases related to aging. Recent studies of apoptosis have impliedthat a common metabolic pathway leading to cell death may be initiatedby a wide variety of signals, including hormones, serum growth factordeprivation, chemotherapeutic agents, and ionizing radiation. Wyllie(1980) Nature, 284:555-556; Kanter et al. (1984) Biochem. Biophys. Res.Commun., 118:392-3999; Duke and Cohen (1986) Lymphokine Res., 5:289-299;Tomei et al. (1988) Biochem. Biophys. Res. Commun., 155:324-331; andKruman et al. (1991) J. Cell. Physiol., 148:267-273.

Although agents that affect apoptosis have therapeutic utility in a widevariety of conditions, it has not been possible to screen for theseagents based on their apoptotic modulating activity. Such assays requirea cell strain that can be maintained in vitro and retain sensitivity toapoptosis modulating signals. The vast majority of cell lines used toscreen agents are selected for their ability to be maintained in vitro.Cells that are most easily maintained are "transformed" cells that havelost the ability to undergo apoptosis and are thus unsuitable for use inscreening apoptosis modulating agents. Although cell strains such as themouse embryonic C3H-10T1/2 type have been shown to be sensitive to suchagents, it has not been possible to perpetuate a phenotypically stablestrain of these cells beyond 120-140 population doublings. Thus, it hasbeen impossible to obtain the reproducible results necessary to engineera high through-put replicate culture assay for drug screening.

It has now been found that, by the method described herein, cell strainssensitive to apoptotic agents can be used to provide reproducibleresults in screening for agents that modulate a wide variety ofdisorders. These phenotypically stable cell strains can now bemaintained for at least 900 population doublings. Moreover, by utilizingthe parameters of the screening assay, new, improved cell strains cannow be obtained that are suitable for use in the screening assays andfor studying apoptosis.

SUMMARY OF THE INVENTION

The present invention is directed to a method of screening agents forpotential therapeutic efficacy. The method comprises culturing a cellstrain that is sensitive to apoptotic modulating agents for a period oftime and under conditions sufficient to attain exponential proliferationactivity without density-dependent constraints at any time during theassay. The cell strain is then exposed to conditions that are known toinduce apoptosis and either simultaneously or shortly thereafter exposedto an agent to be screened. The cell strain is cultured for a time andunder conditions suitable to attain a density of about one populationdoubling from density-dependent arrest of cell cycle. Cells that displaydiminished adherence are then removed and enumerated. The remainingadherent cells are then incubated in the presence of a proteinase for atime and under conditions suitable to release proteinase sensitive cellsfrom the adhesion surface. The proteinase sensitive cells are removedand enumerated to yield the remaining proteinase resistant adherentcells. The proteinase resistant cells are then incubated for a time andunder conditions suitable to release the cells. The released cells arecollected and enumerated.

It is possible that treatment of certain diseases requires thattherapeutic agents either specifically induce apoptosis in instanceswhere deletion of cells is believed to be therapeutically effective suchas in the treatment of cancer. Inhibition of apoptosis is preferred ininstances where prevention of cell loss is believed to betherapeutically effective such as in prevention of ionizingradiation-induced cell death in the gastrointestinal system. The agentis determined to have potential anti-apoptotic therapeutic efficacy ifthe number of non-adherent cells is reduced relative to the number ofnon-adherent cells in the control, and the number of proteinaseresistant cells increases relative to a control. The agent is determinedto have apoptosis inducing therapeutic efficacy if there is an increasein the number of non-adherent cells relative to the number ofnon-adherent cells in the control.

The method can also be used to obtain and maintain phenotypically stablecell strains suitable for use in the screening assay or any other assayrequiring cells that are responsive to apoptosis inducing and apoptosisinhibiting agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of the cell culture method described in Example1.

FIG. 2 is a bar graph depicting apoptotic/trypsin sensitive/trypsinresistant cell count analysis at 24 hours post serum-deprivation. FIG. 2is discussed in Example 3.

FIG. 3 is a bar graph depicting apoptotic/trypsin sensitive/trypsinresistant cell count analysis at 48 hours post serum-deprivation. FIG. 3is discussed in Example 3.

FIG. 4 is a bar graph depicting apoptotic/trypsin sensitive/trypsinresistant cell count analysis at 24 hours post serum-deprivation ofSF90, alanate 166, and alanate 180. FIG. 4 is discussed in Example 3.

DETAILED DESCRIPTION OF THE INVENTION

1. Screening Assay

The present invention is to a method of screening agents for potentialtherapeutic efficacy. The method comprises providing a cell strain thatis sensitive to apoptotic agents; culturing the cell strain for a timeand under conditions sufficient to attain exponential proliferationwithout density dependent constraints at any time during the assay;exposing the cell strain to conditions that are known to induceapoptosis; exposing the cell strain to an agent to be screened;culturing the cell strain for a time and under conditions suitable toattain a density of about one population doubling from density-dependentarrest of cell cycle; removing and enumerating cells that displaydiminished adherence. The remaining adherent cells are then incubated inthe presence of a proteinase for a time and under conditions suitable torelease proteinase sensitive cells from the adhesion surface. Theproteinase sensitive cells are removed and enumerated to yield theremaining proteinase resistant adherent cells. The proteinase resistantcells are then incubated for a time and under conditions suitable torelease the cells. The released cells are collected and enumerated.

The agent is determined to have potential therapeutic efficacy if thenumber of non-adherent cells is reduced relative to the number ofnon-adherent cells in the control, and the number of proteinaseresistant cells increases relative to a control.

The cell strain is obtained from pluripotent embryonic cells withstable, normal, intact, phenotypes. The cell strain is an adherentstrain, its in vitro growth is dependent on anchorage to the solidsupport on which it is grown.

Preferably, the cell strain has the following characteristics: densitydependent regulation of proliferation and death; pluripotent and capableof demonstrating at least two distinct differentiated cell types uponsuitable stimulus; sensitive to loss due to transformation induced bycarcinogenic and oncogenic agents such as DNA damaging agents or variousoncogenic viruses; response to agents that both induce and blockproliferation, differentiation, and apoptosis; ability to exhibitapoptotic cell death as marked by ultrastructural changes of chromatinand specific changes in the molecular structure of DNA associated withapoptotic DNA degradation. More preferably the cell strain is the mouseembryonic C3H-10T1/2 clone 8. This cell strain is available from theAmerican Type Culture Collection under accession number ATCC CCL 226 andis designated C3H-10T1/2, clone 8. C3H10T1/2 has also ben described foruse in transformation experiments. Yavelow et al. (1985) Proc. Natl.Acad. Sci. USA, 82:5395-5399.

The initial culturing step is to attain stable exponential proliferationwithout density dependent constraints at any time during the assay. Inthe case of C3H-10T1/2, this is for about 5 days at 37° C. with a gasoverlay of 5% CO₂ in a suitable growth medium. Although the optimalmedium will vary depending on the cell strain used, in the case ofC3H-10T1/2, the preferred medium is Eagle's basal medium with Hank'ssalts (BME) supplemented with 10% (v/v) heat-inactivated fetal bovineserum. In the case of C3H-10T1/2, after five days of incubation, thecells have reached about seventy-five percent confluency. Although thisis the preferred confluency, particularly for C3H-10T1/2, the confluencycan be greater or lesser so long as statistically significant resultsare obtained. Although the optimal confluency for each cell strain isderived empirically, this is a straightforward determination based onthe parameters discussed below.

Incubating the cells so that they maintain exponential proliferationwithout density dependent constraints during the assay preventsartificial skewing of the results. This is because once the cells reacha certain density, exponential proliferation ceases and the cells becomequiescent. The density at which quiescence occurs is unique to each cellstrain and is empirically derived by measuring indicia of cell divisionsuch as manufacture of DNA. Quiescent cells do not respond to apoptoticand anti-apoptotic agents and thus provide erroneous results.

The level of confluency reached before adding the apoptotic agentdepends only on maintenance of the exponential growth phase andattaining a statistically significant number of cells. In the case ofC3H-10T1/2, seventy-five percent confluency is preferred. The optimalconfluency for any strain is empirically derived.

The condition that induces apoptosis in the cells after the initialincubation step includes but is not limited to serum deprivation, growthfactor deprivation, ultraviolet radiation, γ-radiation, softβ-radiation, hypo-osmotic shock, chemotherapeutic agents, or specificreceptor mediated agents. In the case of C3H-10T1/2, the preferredmethod of inducing apoptosis in a reproducible portion of the cellstrain in replicate cultures is to replace the serum-supplemented growthmedium with serum-free growth medium. After the change to serum-freemedium, the cells are incubated for a time and under conditions suitableto maintain exponential proliferation without density dependentconstraints in suitable replicate control cultures. In the case ofC3H-10T1/2, the conditions are for about 20 hours at 37° C. in anatmosphere of 5% CO₂.

Culturing of cells is dependent upon fetal bovine serum supplement tothe synthetic growth medium. It is important to preserve both the growthpromoting potential of serum supplements as well as the sensitivity toapoptosis inducers. It has now been found that serum production lotsvary not only with respect to proliferative potential, but also withrespect to the ability of the serum to promote apoptosis responsivenessin the cells. This aspect of the assay is counter-intuitive in as muchas conventional serum screening tests used in the art are basedprimarily upon determining the relative ability of various productionbatches of animal sera to maintain high viability or survival rates ofvarious reference cell cultures. Contrary to that rationale, theapoptosis assay serum screening test determines the relative ability ofvarious serum production batches to yield apoptotic, or dying and deadcells upon withdrawal of the growth medium. Thus, each serum lot must beselected from a screened group of several lots in order to ensurepreservation of the cell strain responsiveness to both apoptosisinducers and inhibitors.

The agents to be assayed are introduced to the cells eithersimultaneously with or shortly after induction of apoptosis. Althoughthe time of introduction will vary according to the cell strain used andthe method of induction of apoptosis, the optimal parameters can bedetermined empirically and applied in a routine basis thereafter. In thecase of C3H-10T1/2, the agent to be assayed is added simultaneously withinduction of apoptosis.

After addition of the putative therapeutic agent, the cells are furtherincubated for a time sufficient to display apoptosis. At that time, acertain proportion of the cells in each replicate culture displayrounding and loss of flattened shape, diminished adherence ann areremoved by a mild shearing effected by washing with a suitable bufferedsalt solution.

After the nonadherent cells are removed and enumerated, the remainingadherent cells are comprised of proteinase sensitive and proteinaseresistant cells. It has now been found that the proteinase sensitivecells are the population that contains the cells that would haveotherwise died by apoptosis in the absence of an effective apoptosisinhibitor. The proteinase sensitive cells are removed for enumeration byincubation with a suitable proteinase. A suitable proteinase is trypsin.Trypsin is the standard proteinase for removing adherent cells; however,any proteinase that effectively removes proteinase sensitive cellswithout damaging the cells removed or the remaining proteinase resistantcells is suitable for use in the present invention.

In the case of C3H-10T1/2, the adherent cells are incubated in thepresence of concentrations of about 1 mg/ml to 1 μg/ml trypsin for about10 minutes with gentle shaking at room temperature and are removed bywashing. The proteinase sensitive cells are then enumerated by asuitable method.

Suitable enumeration methods include but are not limited to directphysical counting, electronic particle counting and fluorescencestaining to measure DNA. Any cell counting means known in the art issuitable for use in the present invention.

The proteinase resistant cells are then released. Preferably, release isaccomplished by incubation in the presence of an agent that decreasesthe calcium concentration to a level sufficient to release the cells.Such agents include but are not limited to calcium chelators such asethylenediaminetetraacetic acid and ethylene glycol-bis(β-amino-ethylether) N,N,N',N'-tetraacetic acid.

The number of agents suitable for assaying for therapeutic effectivenessis virtually limitless and can be obtained from a variety of chemical,nutritional and biological sources. For instance, suitable chemicalagents can be novel, untested chemicals, as well as agonists,antagonists, or modifications of known therapeutic agents. Nutritionalagents can be complex extracts from plant or animal sources or extractsthereof. Such agents can be easily derived from plant sources such assoy, pea, or potato by-products, or from animal products such as whey orother milk byproducts. Biological agents include biological responsemodifiers, antibodies and other small molecules. Such small moleculescan be either derived from biological sources or chemically synthesizedto mimic the effect of biological response modifiers.

The assay is suitable for use in a high throughput format, thus enablingthe screening of large numbers of agents in a range of concentrations.Most therapeutic agents screened to date have been effective in aconcentration of about 1 μg/ml, although a wide range of concentrationscan be screened. Preferably, the range of concentrations is from 1 ng/mlto 1 mg/ml. The upper range is limited by solubility of the agent andthe lower range is not necessarily limited but should be in atherapeutically effective range.

The assay is preferably performed in standard tissue culture 60 mm Petridishes which have about 20 cm² surface area. Although the assay may bescaled down to 2 cm² tissue culture dishes, it is not suitable for usein microtiter plates having significantly lower growth surface areas.The cell strain must be maintained as a spatially random distributedpopulation that can not be maintained in the small surface area of amicrotiter dish.

A positive response to the agents is clear. Typically the agents foundto have therapeutic efficacy by decreasing apoptosis have been found toincrease proteinase resistant cells in a statistically significantmanner. These agents also statistically reduce the number ofnon-adherent, apoptotic cells.

It has also now been shown that agents that reduce the number ofnon-adherent cells may do so through a non-specific action unrelated toreduction of apoptosis. Thus, merely measuring the number ofnon-adherent cells that appear in replicate treated cultures is subjectto error in the form of false positive results and is insufficient forthe screening assay. For instance, toxic agents that may rapidly damagecells and lead to disintegration of the cells may be falsely determinedto reduce the appearance of apoptotic cells by virtue of the reductionin non-adherent cell counts. This has now been found to be an avoidableerror if proteinase resistant cells are simultaneously determined in thereplicate cultures. Also, enumeration of a proteinase resistant cellnumber is inherently more reliable than measurement of a loss of cellsby a subtraction method.

The cell strain utilized in the assay is particularly sensitive toapoptosis inducers and inhibitors and is therefore useful in identifyingagents with therapeutic efficacy in treating diseases and conditionsrelated to aging and cellular differentiation. These conditions anddiseases include but are not limited to cardiovascular disease, cancer,immunoregulation, viral diseases, anemia, neurological disorders,diarrhea and dysentery, diabetes, hair loss, rejection of allografts,prostrate hypertrophy, obesity, ocular disorders, stress, and the agingprocess itself.

The assay is not limited to diseases and conditions related to aging,however, as the list above indicates, many conditions associated withaging may also be the result of illness or other physical insults. Forinstance, while not related to aging per se, the trauma induced by headand spinal cord injuries results in apoptotic events that cause thesubsequent cellular loss and the consequent exacerbation of paralysis inmany patients. Agents useful in preventing apoptosis will be useful inminimizing such paralysis.

It has now been found that the assay further distinguishes betweenagents that have therapeutic utility and those that block apoptosis butare toxic. By comparing the proportion of apoptotic, proteinasesensitive and proteinase resistant cells, the assay detects those agentsthat modulate proteinase sensitivity (the apoptosis marker) rather thanthose agents that merely decrease the number of nonadherent (dead)cells.

The assay further provides the means to determine effects of agents onproliferation such as those which have mitogenic activity in addition toantiapoptotic activity.

2. Method of Obtaining Suitable Cell Strains for the Screening Assay

The invention further provides a method of obtaining cell strains otherthan C3H-10T1/2 that retain responsiveness to apoptosis modulatingagents. The method, similar to that above, comprises obtaining a cellsample; culturing the cell sample for a time and under conditionssufficient to maintain exponential proliferation without densitydependent constraints; exposing the cell sample to conditions thatinduce apoptosis; treating the cell sample with an agent known to induceproteinase resistance; culturing the cell sample for a time and underconditions suitable to attain a density of about one population doublingfrom density-dependent arrest of cell cycle; removing cells that displaydiminished adherence to yield adherent cells; exposing the adherentcells to a proteinase; incubating the adherent cells for a time andunder conditions suitable to release proteinase sensitive cells; andcollecting the remaining proteinase resistant cells. These proteaseresistant cells are then retained and passaged to generate a stable cellstrain that continues to express responsiveness to apoptosis inducersand inhibitors.

The preferred treatment of cells for maintenance of apoptosisresponsiveness is 12-O-tetradecanoyl phorbol-13-acetate (TPA) at aconcentration of about 10⁻⁹ to 10⁻⁸ molar and commercially availablefrom Sigma Chemical Company and others.

The incubation conditions and other parameters are essentially similaras those described for the assay described above.

The cell sample is preferably obtained from pluripotent embryonic cellswith a normal, intact, phenotype. The species the cell sample is derivedfrom is not as important as the embryonic origin of the cells. This isbecause embryonic cells are controlled by highly conserved, commonproteins that exhibit minimal species specific variations important toadult organisms. The cell strain should not be transformed, that is,should not be a so-called "immortalized" or tumorigenic cell line. Suchimmortal cell lines have lost most or all of their ability to respond toapoptosis modulating signals or agents.

3. Method of Passaging the Cell Strain

The invention further provides a method of passaging the cell strains soas to preserve phenotypic fidelity defined in part but not completely bycellular responsiveness to apoptosis modulating agents. The methodcomprises culturing a cell strain that is sensitive to apoptoticmodulating agents for a time and under conditions sufficient to attainexponential proliferation activity without density-dependent constraintsat any time during the method. The cell strain is then exposed toconditions that are known to induce apoptosis. The cell strain iscultured for a time under conditions suitable to attain a density ofabout one population doubling from density-dependent arrest of cellcycle. Cells are then removed that display diminished adherence andenumerated. The remaining adherent cells are then incubated in thepresence of a proteinase for a time and under conditions suitable torelease proteinase sensitive cells from the adhesion surface. Theproteinase sensitive cells are removed and enumerated to yield theremaining proteinase resistant adherent cells. The proteinase resistantcells are then incubated for a time and under conditions suitable torelease the cells. The released cells are collected and enumerated.

The incubation conditions and other parameters are essentially similaras those described for the assay described above.

The following examples are provided to illustrate but not limit theclaimed invention. The examples describe the identification oftherapeutically effective agents by the use of the present invention.

EXAMPLES Example 1 Cell Culture Techniques

FIG. 1 depicts a flow chart of the basic technique of cell culture forthe assay. The following is a more detailed description of the assay asperformed using C3H-10T1/2 clone 8 mouse embryonic cells.

The cells are obtained at the lowest available serial passage levelpreferably less than level 11. The phenotypic characteristics of thecells are verified as meeting the following criteria. Standard cellculture techniques are used.

1. Confirmation of a mean doubling time of 22 (±2) hours under standardconditions of basal Eagle's growth medium supplemented with 10% (v/v)fetal bovine serum in plastic culture flasks maintained at 37° C. in ahumidified atmosphere of 5% CO₂.

2. Cloning efficiency determined to be 25% (±2) at densities of 200cells/20 cm² under standard growth conditions.

3. Saturation cell density confirmed to be 5×10⁵ (±2×10⁵) cells/20 cm²plastic petri dish under standard growth conditions.

4. At saturation cell density, it is confirmed that more than 98% ofcells are in the G₁ phase of the cell cycle.

5. The morphology of exponentially proliferating cultures is radicallychanged at saturation density such that the spindle shaped cells havingextensive overlapping and lack of parallel orientation duringexponential proliferation changes to wide, flat epithelioid monolayerwithout distinct intercellular demarcation and no overlapping.

6. Cells are sensitive to malignant transformation by chemicalcarcinogens, typically 3-methyl cholanthrene, or ultraviolet irradiationyielding transformed foci.

7. Cells do not form a fibrosarcoma tumor when injected at levels of 10⁵cells subcutaneously in the suprascapular region of syngeneic animals,whereas, following malignant transformation in vitro, tumors areobserved under similar conditions.

The cells are then cultured in containers that are typically 60 mmdiameter plastic petri dishes specially prepared for mammalian cellculture and are commonly available from several commercial sources.

Cells for replicate culture are obtained from stock cultures which areconfirmed to be in exponential phase proliferation and not inpost-confluent saturation density to ensure that cells-are not arrestedin the G₁ phase of the cell cycle.

The cells are seeded onto each plate in a volume of 5 ml complete growthmedium in which are suspended a standardized number of cells. Thestandardized number should be not less than 10³ but not greater than 10⁴cells.

Special care must be taken to ensure that cells are uniformlydistributed over the surface of each culture dish in order to preventclustering of cells during subsequent growth which results innon-uniform increases in cell densities and premature saturationdensities in limited regions on each dish. Premature saturation densityin limited regions results in serious errors in assay results.

Growth medium is renewed each 48 hours.

When cell density reaches approximately 70% uniformly across each dishsurface, which typically corresponds to a density of 1×10⁵ to 3×10⁵cells/dish, the complete growth medium is removed by aspiration andreplaced with fresh serum-free growth medium.

Drugs or agents to be assayed can be premixed into the serum-freemedium, or added in appropriately small volumes immediately after themedium change.

Typically, in order to ensure statistical reliability, each agent orspecific treatment is performed on at least four replicate cultures andappropriate controls are also incorporated.

After a standardized period of incubation at 37° C. under humidified 5%CO₂ atmosphere of between 3 hours and 72 hours, typically 24 hours, eachplate is prepared for measurement of responses.

The following measurements are performed:

1. All non-adherent or loosely adherent cells are removed from theculture dish and measured by appropriate techniques typically countingby electric particle counting instrument.

2. The remaining adherent cells are exposed to a buffered (typically pH7.3) balanced salt solution such as Hanks Balanced Salt Solutioncontaining a standardized concentration of the enzyme trypsin. Thetrypsin concentration is typically 0.1 mg/ml but can be between 1 and0.001 mg/ml, typically in a volume of 1 ml.

Each culture is incubated either at ambient temperature or 37° C. on arocking platform to ensure uniform distribution of the trypsin reagentover the culture surface. After a standardized period of typically 10minutes, the released cells are removed from each dish, and measured bythe same means described above, typically electronic particle counting.This measurement is referred to as the serum deprivation released or SDRcount and typically contains at least 98% apoptotic cells.

The remaining adherent cells in each dish are then released by exposureto a buffered solution containing a calcium ion chelating agenttypically EDTA typically at a concentration of 2 mg/ml. This measurementis referred to as the proteinase sensitive or PS count and typicallycontains the cells that would have otherwise died by apoptosis in theabsence of an effective inhibitor.

The final cells remaining adhered to the solid support are thenimmediately dispersed and removed from the dish for measurement by thesame means used in previous measurements, typically electronic particlecounting. This measurement is referred to as the proteinase resistant orPR count and typically contains cells that express the property ofresistance to proteinase-induced shape change which has been identifiedto be a critical specific expression related to control of apoptosis.

Each cell count is typically performed in duplicate on each of fourreplicate dishes for each experimental treatment group and control. Thestatistical significance of this is shown in Example 4.

Final data are initially expressed as the mean cell number and standarddeviation of the mean according to conventional statisticaldata-analysis as discussed in Example 4. The results obtained are shownin Table 1. The numbers in Table 1 printed in bold print represent thenumbers used in Table 2 as described in Example 2. In Table 1, the dataare reported as cells/plate×10,000. The abbreviations used are: SD,standard deviation; PR, proteinase resistant cells; and PS, proteinasesensitive.

                                      TABLE 1                                     __________________________________________________________________________    Bovine serum screening test C3H-10T1/2 cells                                          Apoptotic                                                                           +/-SD                                                                              % control                                                                           PR +/-SD                                                                              % control                                                                           PS +/-SD                                                                              % control                                                                           Total                                                                             %                    __________________________________________________________________________                                                             control              Lot #1 Control                                                                        22.6  2.6  100.0 17.7                                                                             3.6  100.0 20.4                                                                             3.3  100.0 60.7                                                                              100.0                1 nM TPA                                                                              19.8  0.6  87.7  27.8                                                                             4.4  157.1 16.7                                                                             2.6  81.9  64.3                                                                              105.9                10 nM TPA                                                                             13.1  1.5  58.0  48.1                                                                             4.4  271.8 15.9                                                                             1.2  77.9  77.1                                                                              127.0                Lot #2 Control                                                                        13.6  0.5  100.0 17.2                                                                             2.1  100.0 20.2                                                                             1.8  100.0 51.0                                                                              100.0                1 nM TPA                                                                              10.4  1.6  76.5  23.5                                                                             3.3  136.6 15.0                                                                             1.8  74.3  48.9                                                                              95.9                 10 nM TPA                                                                             6.0   0.4  44.1  35.6                                                                             2.7  207.0 14.2                                                                             1.3  70.3  55.8                                                                              109.4                Lot #3 Control                                                                        30.8  2.0  100.0 16.7                                                                             5.1  100.0 25.9                                                                             5.2  100.0 73.4                                                                              100.0                1 nM TPA                                                                              25.4  1.3  82.5  26.3                                                                             4.5  157.5 22.3                                                                             2.8  86.1  74.0                                                                              100.8                10 nM TPA                                                                             15.4  1.5  50.0  49.5                                                                             1.8  296.4 13.3                                                                             1.2  51.4  78.1                                                                              106.4                Lot #4 Control                                                                        35.9  1.9  100.0 20.5                                                                             1.0  100.0 34.6                                                                             0.9  100.0 90.9                                                                              100.0                10 nM TPA                                                                             21.6  4.9  60.2  42.6                                                                             6.5  207.8 45.0                                                                             3.4  130.1 110.1                                                                             121.1                50 nM TPA                                                                             16.8  1.4  46.8  47.4                                                                             4.2  231.2 45.9                                                                             4.1  132.7 109.1                                                                             120.0                Lot #5 Control                                                                        49.1  6.0  100.0 20.5                                                                             4.2  100.0 49.6                                                                             2.5  100.0 119.2                                                                             100.0                10 nM TPA                                                                             30.7  5.1  62.5  53.9                                                                             2.5  262.9 47.0                                                                             4.9  94.8  131.6                                                                             110.4                50 nM TPA                                                                             16.7  2.4  34.0  55.6                                                                             6.7  271.2 52.2                                                                             3.2  105.2 124.5                                                                             104.3                __________________________________________________________________________

Initial data-analyses for assay reliability should reveal thatvariations among replicate culture dishes for any of the threemeasurements should not be greater than 7% standard deviation. Data canbe normalized by expressing the individual counts of SDR, PS, and PR asratios to the corresponding value determined in control dishes.

A preferred positive result is typically dependent upon a statisticallysignificant reduction of SDR cells in combination with a statisticallysignificant increase in PR cells. However, putative apoptosis modulatingagents can produce either reduction of SDR cells or increased PR cellsand should be considered to be positive and warranting furtherconsideration. Agents that produce either increased SDR or decreasedtotal cell counts (i.e., SDR+PS+PR) should be considered to bepotentially cytotoxic at the concentrations applied. A negative outcomewould be failure to observe changes in either SDR or PR counts atconcentrations determined to be non-toxic.

Example 2 Bovine Serum Screening Test

The purpose of screening the bovine serum used as a supplement of thesynthetic portion of the cell culture growth medium is to determine thebest manufacturer's production batch in terms of optimal assayperformance. This aspect of the assay is counter-intuitive in as much asconventional serum screening tests used by those skilled in the art arebased primarily on determining the relative ability of variousproduction batches of animal sera to maintain high viability or survivalrates of various reference cell cultures. Contrary to that rationale,the apoptosis assay serum screening test determines the relative abilityof various serum production batches to yield apoptotic, or dying anddead, cells upon withdrawal from the growth medium.

As an example, Tables 1 and 2 present typical data obtained in theprocess of screening 5 different bovine serum production batchesobtained from a commercial source. These are listed as Lots 1 through 5.In order to distinguish between necrotic and apoptotic cells, cells aretreated with TPA. Two independent variables to be measured are thenumber of apoptotic cells in the untreated control cultures comparedwith replicate cultures treated with TPA. These variables are thereleased cell or Apoptotic count, and the proteinase-resistant or PRcell count as shown in Table 1.

Results were evaluated as follows: Apoptotic and PR responses areexpressed in terms of a ratio or percentage of the correspondingresponse obtained in untreated replicate cultures. A desirable responsewould be simultaneous maximal reduction of Apoptotic cell count andenhancement of PR cell count. Therefore, the responses are then rankedaccording to each lot's respective response for each variable as shownin Table 2.

                  TABLE 2                                                         ______________________________________                                        Analysis of results of screening data                                         Activity determined at 10 nM TPA                                              serum            Suppression     Induction                                                                            total                                 lot #    rank    of apoptosis                                                                            rank  of prot-R                                                                            ranking                               ______________________________________                                             1       3       58.0    2     272    5                                        2       1       44.1    5     207    6                                   X    3       2       50.0    1     296    3                                        4       4       60.2    4     208    8                                        5       5       62.5    3     263    8                                   ______________________________________                                    

The results shown in Table 2 indicate that Lot #3 ranked second insuppression of apoptotic cells, and first in induction of trypsinresistance. This sum of 3 was then determined to be the best of the fivelots evaluated and it was then selected for use in the peptide assaydescribed in Example 3.

Example 3 Assay of Putative Therapeutic Peptide Mixtures

In order to determine whether the assay described in Example 1 waseffective in recognizing agents with potential therapeutic activity, thefollowing assay was performed utilizing peptide mixtures. The assay wasperformed essentially as described in Example 1 with C3H-10T1/2 clone 8with the addition of various peptide mixtures. The peptides utilizedwere obtained from soy beans and designated pp/g, EDI, ARD, FXP, GS90FB,pp750, SPH, PRO (commercially available from such sources as ArthurDaniels Midland and Purina); and a protein product obtained from peasand designated PEA commercially available from such companies asGrinsted and Novo Nordisk.

The cells were used during exponential growth phase when cell cycleposition is randomly distributed with no cells arrested in G₀. At T=0,cultures are transferred to serum-free medium as an apoptosis stimulus.Controls included 1 nM TPA to ensure responsiveness of cell cultures,and acetone as a nonspecific solvent control.

Peptide mixtures were added to serum-free medium at the highestconcentration previously determined to exhibit no toxicity as determinedby suppression of cloning efficiency in a conventional cytotoxicityassay. Toxicity was determined by detection of a statisticallysignificant suppression of cell cloning efficiency when the agents wereadded to cell cultures 24 h after seeding at 200 cells per 20 squarecentimeter culture plate. Assays were performed on 4 replicate cultureplates and consisted of counting the number of clones of greater than 16cells at day 7.

Only three peptide mixtures were found to be toxic at 1:10 dilutionlevels of the saturated stock solutions: pp750; SPH; and PRO. At 1:100and 1:1000 dilution levels, none of the peptides were found to be toxic.Analyses of cell responses are made at either 24 hours (FIG. 2 and Table3) or 48 hours (FIG. 3 and Table 4) after serum deprivation.

Three measurements were performed on each culture plate consisting ofdifferential cell counts:

1. Apoptotic cell count: These released cells consist of >95% apoptoticcells as shown by both ultrastructure analysis and DNA fragmentationanalysis.

2. Proteinase resistant cell count: The fraction of cells that areresistant to proteinase-release is counted. These cells represent thesubpopulation that responds to the apoptosis inhibitor TPA and containsthe mitogenic responsive cells. Research indicates that this populationis critical to control of apoptosis, probably through mechanisms relatedto modulation of gene expression through mechanisms related to cellshape and adhesion to substrates. This response has been shown to berelated but independent of simple suppression of apoptotic cell number.It remains a critical but empirical marker for apoptosis modulation.

3. Proteinase-sensitive cell count: This is the remaining fraction ofthe total cell population which exhibits high sensitivity to proteinaserelease from adhesion surface. At present this count is used tocalculate the total cell number in each plate in order to determinewhether the agent had mitogenic properties.

The results obtained are shown in FIGS. 2 and 3 Tables 3 and 4.

                                      TABLE 3                                     __________________________________________________________________________    Experiment 158                                                                Effects Determined at 24 hours                                                    medium                                                                              solvent                                                             (SD)                                                                              control                                                                             control                                                                             TPA 1 nM                                                                            sph   pro   g590-fb                                                                             pea                                   __________________________________________________________________________    Apo 126.8(15.7)                                                                         114.2(23.1)                                                                         85.9(11.0)                                                                          130.9(15.7)                                                                          90.9(18.2)                                                                         122.5(27.0)                                                                          78.0(11.2)                           PS  254.7(11.6)                                                                         253.3(2.6)                                                                          271.6(17.4)                                                                         222.2(31.7)                                                                         218.2(29.2)                                                                         196.9(25.9)                                                                         63.3(6.0)                             PR  39.2(4.8)                                                                           36.2(4.0)                                                                           99.0(33.5)                                                                          66.0(5.9)                                                                           83.7(4.5)                                                                            83.7(16.2)                                                                         261.5(12.0)                           Total                                                                             420.7 403.7 456.5 419.1 392.8 403.1 402.8                                 __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Experiment 158                                                                Effects determined at 48 hours                                                meduim   solvent                                                                           TPA 1 nM                                                                            sph                                                                              pro                                                                              g590-fb                                                                           pea                                                                              sph(+)                                                                            pro(+)                                                                            pea(+)                                                                            g590-fb(+)                        __________________________________________________________________________    Apo 126.4                                                                              105.1                                                                             54.1  100.2                                                                            79.2                                                                             34.8                                                                              141.3                                                                            89.8                                                                              62.1                                                                              103.2                                                                             58.2                              PS  150.2                                                                              115.2                                                                             43.3  136.6                                                                            118.4                                                                            112.7                                                                             29.8                                                                             58.4                                                                              59.2                                                                              24.8                                                                              45.8                              PR  92.6 105.8                                                                             306.7 72.6                                                                             96.1                                                                             103.6                                                                             178.4                                                                            242.2                                                                             232.8                                                                             240.7                                                                             248.4                             Total                                                                             369.1                                                                              326.1                                                                             404.1 309.4                                                                            293.7                                                                            251.1                                                                             349.5                                                                            390.4                                                                             354.1                                                                             368.7                                                                             352.4                             __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    Experiment 163                                                                                     Alanate-166                                                                         Alante-166                                                                          Alante-166                                   control    acetone                                                                            SE90F                                                                              1:1000                                                                              1:1000                                                                              1:100 TPA                                    __________________________________________________________________________    Apo   14/1(1.1)                                                                          15.3(1.5)                                                                          10.8(2.3)                                                                           4.6(0.4)                                                                            9.2(0.7)                                                                            6.3(0.4)                                                                           21.7(0.7)                              PS    27.1(1.5)                                                                          17.8(1.3)                                                                          27.8(8.5)                                                                          25.4(4.7)                                                                           22.3(5.2)                                                                           29.4(5.1)                                                                           19.6(2.3)                              PR    39.8(3.7)                                                                          46.1(4.5)                                                                          31.3(0.6)                                                                          46.8(5.9)                                                                           43.8(1.7)                                                                           50.2(1.7)                                                                           61.1(5.2)                              Total cells/                                                                        81.3 79.2 69.9 76.8  75.3  85.9  102.4                                  plate                                                                         __________________________________________________________________________

                                      TABLE 6                                     __________________________________________________________________________    Experiment 157: Peptide mixtures in presenence and absence of TPA             Cells per culture plate × 1000                                                                       apo  PR                                          +/-1 nM TPA                                                                            Apo                                                                              PS  PR Total                                                                              mitogenic                                                                          inhibition                                                                         activity                                    __________________________________________________________________________    medium control                                                                         140.1                                                                            30.50                                                                             177.1                                                                            347.7                                                      solvent control                                                                        173.0                                                                            35.15                                                                             187.0                                                                            395.2                                                      TPA 1 nM 125.1                                                                            24.80                                                                             293.4                                                                            443.6                                                                              yes  yes  yes                                         pro(-)   91.3                                                                             35.60                                                                             162.5                                                                            289.4                                                                              no   yes  no                                          pro(+)   63.1                                                                             32.80                                                                             226.4                                                                            322.2                                                      pp750(-) 112.2                                                                            26.60                                                                             189.9                                                                            328.7                                                                              no   yes  no                                          pp750(+) 73.3                                                                             21.40                                                                             210.9                                                                            304.6                                                      g590-fb(-)                                                                             40.2                                                                             36.40                                                                             182.6                                                                            259.1                                                                              no   yes  no                                          g590-fb(+)                                                                             73.4                                                                             27.60                                                                             256.4                                                                            357.4                                                      pea(-)   92.0                                                                             20.40                                                                             182.8                                                                            298.2                                                                              no   yes  no                                          pea(+)   126.2                                                                            24.00                                                                             216.7                                                                            367.0                                                      pp16-10(-)                                                                             228.0                                                                            13.40                                                                             206.0                                                                            447.3                                                                              yes  no   no                                          pp16-10(+)                                                                             187.7                                                                            12.10                                                                             295.3                                                                            497.1                                                      fxp 720(-)                                                                             231.4                                                                            11.10                                                                             196.4                                                                            438.9                                                                              yes  no   no                                          fxp 720(+)                                                                             204.3                                                                            13.30                                                                             279.3                                                                            496.9                                                      ard(-)   201.9                                                                            16.30                                                                             206.7                                                                            424.9                                                                              yes  no   no                                          ard(+)   180.2                                                                            18.50                                                                             276.7                                                                            478.5                                                      edi(-)   151.5                                                                            19.20                                                                             146.5                                                                            317.2                                                                              no   no   no                                          edi(+)   131.0                                                                            17.60                                                                             205.1                                                                            353.6                                                      __________________________________________________________________________

As determined by the results depicted in Tables 3-6 and FIGS. 2-4, theactivities of the peptide mixtures were as follows:

1. No Activity. Defined as having no appreciable effect on anymeasurement at concentrations of 1:10, 1:100, and 1:1000. Peptidemixtures exhibiting no activity included SE90F and EDI;

2. Anti-apoptotic. Defined as being active at either 24 or 48 hours postserum deprivation in significantly depressing the number of apoptoticcells in each plate. Peptide mixtures exhibiting anti-apoptotic activityincluded: PRO (1:10, 1:100); pp750(1:100); gs90fb (1:10); PEA (1:10);Alanate 166 (1:100, 1:1000); and Alanate 180 (1:100);

3. PR induction. Defined as able to induce significantly increasedproteinase-resistant adhesion. Peptide mixtures exhibiting PR inductioninclude: PEA (1:10); Alanate 180 (1:100); and SPH (1:1000); and

4. Mitogenic: Defined as able to significantly increase the total cellnumber 24 hours after addition to serum-free medium. Peptides exhibitingmitogenic activity include: pp16-10(1:10); fxp-720 (1:10); and ARD(1:10).

It should be noted that only PEA and alanate-180 were found to haveactivity in two measurements, Anti-apoptotic activity and PR inductionactivity. Although neither was mitogenic, PEA also did not interferewith TPA mitogenicity when added in combination.

It has also been found that agents may effect apoptosis at 24 or 48hours. Consequently, it is preferable to analyze the effect of putativetherapeutic agents at both 24 and 48 hours.

Example 4 Statistical Analysis of Screening Assay

In order to determine the requisite sample size to assure statisticallysignificant results, the results presented in Tables 3 and 4 wererecompiled and subject to statistical analysis. The statistical analysiswas performed as described in Daniel, Biostatistics: A Foundation in theHealth Sciences, 2 ed. John Wiley and Sons (1978). The results of theassessment of statistical significance of sample size are presented inTab1e 7.

Typical results were used including mean values and standard deviationof the means from actual assay measurements obtained as previouslydescribed by Daniel (1978). Mean values of both control andpeptide-treated groups and their respective standard deviations aretabulated followed by sample sizes of either 2 or 4 replicate cultureplates. Based upon choices of culture replicate values of either 2 or 4,statistics were calculated and levels of significance were determined.The levels of significance were evaluated by selecting the lowest valueof replicates that provided statistical reliability as determined by pvalues of 0.050 or less in order to provide general statisticalreliability at the 95% confidence level.

In Table 7, examples 1-12 are results obtained with G590-FB at 24 hours(1-6) and 48 hours (7-12) respectively; examples 13-24 are thoseobtained with TPA at 1 nM at 24 hours (13-18) and 48 hours (19-24)respectively; and examples 25-36 are those obtained with PEA proteins at24 hours (25-30) and 48 hours (31-36) respectively.

                                      TABLE 7                                     __________________________________________________________________________    STATISTICAL ASSESSMENT OF METHODOLOGIES TO IDENTIFY POSSIBLE APOPTOSIS        MODULATING PEPTIDES                                                           SAM-                          EQUAL                                                                              EQUAL                                      PLE               CON-        VARI-                                                                              VARI-                                      CON- PEP- CON-                                                                              PEP-                                                                              TROL  PEPTIDE                                                                             ANCE ANCE POOLED             P-                 TROL TIDE TROL                                                                              TIDE                                                                              SAMPLE                                                                              SAMPLE                                                                              TEST:F                                                                             TEST:P-                                                                            VARI-     T        VAL-               MEAN MEAN SD  SD  SIZE  SIZE  STAT VALUE                                                                              ANCE  TEST                                                                              STAT DF  UE                 __________________________________________________________________________     1. 126.8                                                                          122.5                                                                              15.7                                                                              27  2     2     2.95752                                                                            0.67060                                                                            487.745                                                                             =   0.19470                                                                            2.00000                                                                           0.8636                                                           ≠                                                                           0.19470                                                                            1.60686                                                                           1                                                                             0.8673                                                                        8                   2. 126.8                                                                          122.5                                                                              15.7                                                                              27  4     4     2.95752                                                                            0.39693                                                                            487.745                                                                             =   0.27535                                                                            6.00000                                                                           0.7922                                                           ≠                                                                           0.27535                                                                            4.82058                                                                           8                                                                             0.7944                                                                        6                   3. 254.7                                                                          196.9                                                                              11.6                                                                              25.9                                                                              4     4     4.98521                                                                            0.21988                                                                            402.685                                                                             =   4.07343                                                                            6.00000                                                                           0.0065                                                           ≠                                                                           4.07343                                                                            4.15700                                                                           50                                                                            0.0140                                                                        41                  4. 254.7                                                                          196.9                                                                              11.6                                                                              25.9                                                                              2     2     4.98521                                                                            0.53614                                                                            402.685                                                                             =   2.88035                                                                            2.00000                                                                           0.1023                                                           ≠                                                                           2.88035                                                                            1.38567                                                                           6                                                                             0.1534                                                                        6                   5. 39.2                                                                           83.7 4.8 16.2                                                                              4     4     11.3906                                                                            0.075934                                                                           142.74                                                                              =   -5.26747                                                                           6.00000                                                                           .00188                                                           ≠                                                                           -5.26747                                                                           3.52272                                                                           78                                                                            .00879                                                                        46                  6. 39.2                                                                           83.7 4.8 16.2                                                                              2     2     11.3906                                                                            0.36676                                                                            142.74                                                                              =   -3.72466                                                                           2.00000                                                                           0.0651                                                           ≠                                                                           -3.72466                                                                           1.17424                                                                           2                                                                             0.1373                                                                        6                   7. 126.4                                                                          34.8 15.6505                                                                           7.67020                                                                           4     4     4.16333                                                                            0.27195                                                                            151.885                                                                             =   10.5112                                                                            6.00000                                                                           .00004                                                           ≠                                                                           10.5112                                                                            4.36254                                                                           356                                                                           .00029                                                                        166                 8. 126.4                                                                          34.8 15.6505                                                                           7.67020                                                                           2     2     4.16333                                                                            0.58020                                                                            151.885                                                                             =   7.43256                                                                            2.00000                                                                           0.0176                                                           ≠                                                                           7.43256                                                                            1.45418                                                                           25                                                                            0.0394                                                                        26                  9. 150.2                                                                          112.7                                                                              6.84068                                                                           14.8244                                                                           4     4     4.69632                                                                            0.23614                                                                            133.279                                                                             =   4.59372                                                                            6.00000                                                                           .00371                                                           ≠                                                                           4.59372                                                                            4.22218                                                                           65                                                                            .00886                                                                        37                 10. 150.2                                                                          112.7                                                                              6.84068                                                                           14.8244                                                                           2     2     4.69632                                                                            0.55046                                                                            133.279                                                                             =   3.24825                                                                            2.00000                                                                           0.0831                                                           ≠                                                                           3.24825                                                                            1.40739                                                                           3                                                                             0.1295                                                                        0                  11. 92.5                                                                           103.6                                                                              11.3265                                                                           20.0516                                                                           4     4     3.13404                                                                            0.37322                                                                            285.179                                                                             =   -0.96398                                                                           6.00000                                                                           0.3722                                                           ≠                                                                           -0.96398                                                                           4.73756                                                                           8                                                                             0.3816                                                                        4                  12. 92.5                                                                           103.6                                                                              11.3265                                                                           20.0516                                                                           2     2     3.13404                                                                            0.65468                                                                            265.179                                                                             =   -0.68164                                                                           2.00000                                                                           0.5658                                                           ≠                                                                           -0.68164                                                                           1.57919                                                                           1                                                                             0.5815                                                                        4                  13. 254.7                                                                          271.6                                                                              11.6                                                                              17.4                                                                              2     2     2.25 0.74867                                                                            218.66                                                                              =   -1.14288                                                                           2.00000                                                                           0.3714                                                           ≠                                                                           -1.14288                                                                           1.74227                                                                           5                                                                             0.3859                                                                        9                  14. 254.7                                                                          271.6                                                                              11.6                                                                              17.4                                                                              4     4     2.25 0.52265                                                                            218.66                                                                              =   -1.61628                                                                           6.00000                                                                           0.1571                                                           ≠                                                                           -1.61628                                                                           5.22680                                                                           6                                                                             0.1644                                                                        2                  15. 39.2                                                                           99   4.8 33.5                                                                              2     2     48.7088                                                                            0.18120                                                                            572.645                                                                             =   -2.49896                                                                           2.00000                                                                           0.1297                                                           ≠                                                                           -2.49896                                                                           1.04104                                                                           0                                                                             0.2343                                                                        4                  16. 39.2                                                                           99   4.8 33.5                                                                              4     4     48.7088                                                                            .0096292                                                                           572.645                                                                             =   -3.53406                                                                           6.00000                                                                           0.0123                                                           ≠                                                                           -3.53406                                                                           3.12313                                                                           07                                                                            0.0361                                                                        29                 17. 126.8                                                                          85.9 15.7                                                                              11  2     2     2.03711                                                                            0.77814                                                                            183.745                                                                             =   3.01728                                                                            2.00000                                                                           0.0945                                                           ≠                                                                           3.01728                                                                            1.79114                                                                           3                                                                             0.1078                                                                        9                  18. 126.8                                                                          85.9 15.7                                                                              11  4     4     2.03711                                                                            0.57382                                                                            183.745                                                                             =   4.26708                                                                            6.00000                                                                           .00528                                                           ≠                                                                           4.26708                                                                            5.37342                                                                           00                                                                            .00677                                                                        53                 19. 126.4                                                                          54.1 15.6505                                                                           6.92782                                                                           4     4     5.10342                                                                            0.21376                                                                            146.466                                                                             =   8.44860                                                                            6.00000                                                                           .00015                                                           ≠                                                                           8.44860                                                                            4.13221                                                                           014                                                                           .00093                                                                        096                20. 126.4                                                                          54.1 15.6505                                                                           6.92782                                                                           2     2     5.10342                                                                            0.53060                                                                            146.466                                                                             =   5.97406                                                                            2.00000                                                                           0.0268                                                           ≠                                                                           5.97406                                                                            1.37740                                                                           94                                                                            0.0598                                                                        35                 21. 150.2                                                                          43.3 6.84068                                                                           2.77401                                                                           4     4     6.08111                                                                            0.17235                                                                            27.2450                                                                             =   28.9634                                                                            6.00000                                                                           .00000                                                           ≠                                                                           28.9634                                                                            3.96068                                                                           01122                                                                         .00000                                                                        9255               22. 150.2                                                                          43.3 6.84068                                                                           2.77401                                                                           2     2     6.08111                                                                            0.49052                                                                            27.2450                                                                             =   20.4802                                                                            2.00000                                                                           0.0023                                                           ≠                                                                           20.4802                                                                            1.32023                                                                           76                                                                            0.0129                                                                        57                 23. 92.5                                                                           306.7                                                                              11.3265                                                                           103.782                                                                           4     4     83.9562                                                                            .0043206                                                                           5449.53                                                                             =   -4.10350                                                                           6.00000                                                                           0.0063                                                           ≠                                                                           -4.10350                                                                           3.07146                                                                           32                                                                            0.0250                                                                        32                 24. 92.5                                                                           306.7                                                                              11.3265                                                                           103.782                                                                           2     2     83.9562                                                                            0.13841                                                                            5449.53                                                                             =   -2.90162                                                                           2.00000                                                                           0.1010                                                           ≠                                                                           -2.90162                                                                           1.02382                                                                           8                                                                             0.2065                                                                        8                  25. 126.8                                                                          78   15.7                                                                              11.2                                                                              4     4     1.96500                                                                            0.59304                                                                            185.965                                                                             =   5.06080                                                                            6.00000                                                                           .00230                                                           ≠                                                                           5.06080                                                                            5.42531                                                                           89                                                                            .00309                                                                        06                 26. 126.8                                                                          78   15.7                                                                              11.2                                                                              2     2     1.96500                                                                            0.78896                                                                            185.965                                                                             =   3.57853                                                                            2.00000                                                                           0.0699                                                           ≠                                                                           3.57853                                                                            1.80844                                                                           90                                                                            0.0809                                                                        99                 27. 254.7                                                                          63.3 11.6                                                                              6   4     4     3.73778                                                                            0.30754                                                                            85.28 =   29.3112                                                                            6.00000                                                                           .00000                                                           ≠                                                                           29.3112                                                                            4.49801                                                                           01045                                                                         .00000                                                                        26126              28. 254.7                                                                          63.3 11.6                                                                              6   2     2     3.73778                                                                            0.60778                                                                            85.28 =   20.7261                                                                            2.00000                                                                           .00231                                                           ≠                                                                           20.7261                                                                            1.49934                                                                           98                                                                            .00799                                                                        14                 29. 39.2                                                                           261.5                                                                              4.8 12  4     4     6.25 0.16654                                                                            83.52 =   -34.4001                                                                           6.00000                                                                           .00000                                                           ≠                                                                           -34.4001                                                                           3.93604                                                                           00402                                                                         .00000                                                                        49874              30. 39.2                                                                           261.5                                                                              4.8 12  2     2     6.25 0.48448                                                                            83.52 =   -24.3245                                                                           2.00000                                                                           0.0016                                                           ≠                                                                           -24.3245                                                                           1.31201                                                                           86                                                                            0.0105                                                                        70                 31. 126.4                                                                          141.3                                                                              15.6505                                                                           20.2892                                                                           4     4     1.68065                                                                            0.68022                                                                            328.295                                                                             =   -1.16297                                                                           6.00000                                                                           0.2890                                                           ≠                                                                           -1.16297                                                                           5.63660                                                                           0                                                                             0.2917                                                                        0                  32. 126.4                                                                          141.3                                                                              15.6505                                                                           20.2892                                                                           2     2     1.68065                                                                            0.83657                                                                            328.295                                                                             =   -0.82235                                                                           2.00000                                                                           0.4973                                                           ≠                                                                           -0.82235                                                                           1.87887                                                                           2                                                                             0.5020                                                                        9                  33. 150.2                                                                          29.8 6.84068                                                                           2.82464                                                                           4     4     5.86503                                                                            0.18029                                                                            27.3867                                                                             =   32.5365                                                                            6.00000                                                                           .00000                                                           ≠                                                                           32.5365                                                                            3.99411                                                                           00561                                                                         .00000                                                                        53961              34. 150.2                                                                          29.8 6.84068                                                                           2.82464                                                                           2     2     5.86503                                                                            0.49859                                                                            27.3867                                                                             =   23.0068                                                                            2.00000                                                                           0.0018                                                           ≠                                                                           23.0068                                                                            1.33137                                                                           84                                                                            0.0107                                                                        79                 35. 92.5                                                                           178.4                                                                              11.3265                                                                           8.18662                                                                           4     4     1.91419                                                                            0.60723                                                                            97.6555                                                                             =   -12.2931                                                                           6.00000                                                                           .00001                                                           ≠                                                                           -12.2931                                                                           5.46244                                                                           7658                                                                          .00003                                                                        4574               36. 92.5                                                                           178.4                                                                              11.3265                                                                           8.18662                                                                           2     2     1.91419                                                                            0.79686                                                                            97.6555                                                                             =   -8.69250                                                                           2.00000                                                                           0.0129                                                           ≠                                                                           -8.69250                                                                           1.82081                                                                           78                                                                            0.0171                                                                        60                 __________________________________________________________________________

The results obtained from the statistical analysis indicate that thesample size is critical to obtaining statistically significant results.Changing the sample number from 4 to 2 results in a statisticallysignificant change in both the P and T values. Thus, it is preferredthat a sample size of at least 4 be utilized in order to obtainstatistically significant results.

I claim:
 1. A cell strain that retains responsiveness to apoptosismodulating agents obtained by the method comprising the steps of:a)obtaining a cell sample; b) culturing the cell sample for a time andunder conditions sufficient to attain exponential proliferation withoutdensity dependent constraints; c) exposing the cell sample to conditionsthat induce apoptosis while simultaneously treating the cell sample withan agent known to induce proteinase resistance; d) culturing the cellsample for a time and under conditions suitable to maintain exponentialproliferation without density dependent constraints; e) removing cellsthat display diminished adherence to yield adherent cells; f) exposingthe adherent cells to a proteinase; g) incubating the adherent cells fora time and under conditions suitable to release proteinase sensitivecells and yield proteinase resistant cells; h) collecting the proteinaseresistant cells; and i) retaining the cells of step h) and passaging thecells to generate a stable cell strain that retains responsiveness toapoptosis modulating agents.